Flj20647s as modifiers of the p21 pathway and methods of use

ABSTRACT

Human FLJ20647 genes are identified as modulators of the p21 pathway, and thus are therapeutic targets for disorders associated with defective p21 function. Methods for identifying modulators of p21, comprising screening for agents that modulate the activity of FLJ20647 are provided.

REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional patent applicationNo. 60/411,010 filed Sep. 16, 2002. The contents of the priorapplication are hereby incorporated in their entirety.

BACKGROUND OF THE INVENTION

The p21/CDKN1/WAF1/CIP1 protein (El-Deiry, W. S.; et al. Cell 75:817-825, 1993; Harper, J. W.; et al. Cell 75: 805-816, 1993; Huppi, Ketal. Oncogene 9: 3017-3020, 1994) is a cell cycle control protein thatinhibits cyclin-kinase activity, is tightly regulated at thetranscriptional level by p53, and mediates p53 suppression of tumor cellgrowth. Along with p53, p21 appears to be essential for maintaining theG2 checkpoint in human cells (Bunz, F.; Dutriaux, A.; et al. Science282:1497-1501, 1998). Sequences of P21 are well-conserved throughoutevolution, and have been identified in species as diverse as human(Genbank Identifier 13643057), Drosophila melanogaster (GI# 1684911),Caenorhabditis elegans (GI#4966283), and yeast (GI#2656016).

The ability to manipulate the genomes of model organisms such asDrosophila provides a powerful means to analyze biochemical processesthat, due to significant evolutionary conservation, have directrelevance to more complex vertebrate organisms. Due to a high level ofgene and pathway conservation, the strong similarity of cellularprocesses, and the functional conservation of genes between these modelorganisms and mammals, identification of the involvement of novel genesin particular pathways and their functions in such model organisms candirectly contribute to the understanding of the correlative pathways andmethods of modulating them in mammals (see, for example, Mechler B M etal., 1985 EMBO J. 4:1551-1557; Gateff E. 1982 Adv. Cancer Res. 37:33-74; Watson K L., et al., 1994 J Cell Sci. 18: 19-33; Miklos G L, andRubin G M. 1996 Cell 86:521-529; Wassarman D A, et al., 1995 Curr OpinGen Dev 5: 44-50; and Booth D R. 1999 Cancer Metastasis Rev. 18:261-284). For example, a genetic screen can be carried out in aninvertebrate model organism having underexpression (e.g. knockout) oroverexpression of a gene (referred to as a “genetic entry point”) thatyields a visible phenotype. Additional genes are mutated in a random ortargeted manner. When a gene mutation changes the original phenotypecaused by the mutation in the genetic entry point, the gene isidentified as a “modifier” involved in the same or overlapping pathwayas the genetic entry point. When the genetic entry point is an orthologof a human gene implicated in a disease pathway, such as p21, modifiergenes can be identified that may be attractive candidate targets fornovel therapeutics.

All references cited herein, including patents, patent applications,publications, and sequence information in referenced Genbank identifiernumbers, are incorporated herein in their entireties.

SUMMARY OF THE INVENTION

We have discovered genes that modify the p21 pathway in Drosophila, andidentified their human orthologs, hereinafter referred to as FLJ20647.The invention provides methods for utilizing these p21 modifier genesand polypeptides to identify FLJ20647-modulating agents that arecandidate therapeutic agents that can be used in the treatment ofdisorders associated with defective or impaired p21 function and/orFLJ20647 function. Preferred FLJ20647-modulating agents specificallybind to FLJ20647 polypeptides and restore p21 function. Other preferredFLJ20647-modulating agents are nucleic acid modulators such as antisenseoligomers and RNAi that repress FLJ20647 gene expression or productactivity by, for example, binding to and inhibiting the respectivenucleic acid (i.e. DNA or mRNA).

FLJ20647 modulating agents may be evaluated by any convenient in vitroor in vivo assay for molecular interaction with an FLJ20647 polypeptideor nucleic acid. In one embodiment, candidate FLJ20647 modulating agentsare tested with an assay system comprising a FLJ20647 polypeptide ornucleic acid. Agents that produce a change in the activity of the assaysystem relative to controls are identified as candidate p21 modulatingagents. The assay system may be cell-based or cell-free.FLJ20647-modulating agents include FLJ20647 related proteins (e.g.dominant negative mutants, and biotherapeutics); FLJ20647-specificantibodies; FLJ20647-specific antisense oligomers and other nucleic acidmodulators; and chemical agents that specifically bind to or interactwith FLJ20647 or compete with FLJ20647 binding partner (e.g. by bindingto an FLJ20647 binding partner). In one specific embodiment, a smallmolecule modulator is identified using a binding assay. In specificembodiments, the screening assay system is selected from an apoptosisassay, a cell proliferation assay, an angiogenesis assay, and a hypoxicinduction assay.

In another embodiment, candidate p21 pathway modulating agents arefurther tested using a second assay system that detects changes in thep21 pathway, such as angiogenic, apoptotic, or cell proliferationchanges produced by the originally identified candidate agent or anagent derived from the original agent. The second assay system may usecultured cells or non-human animals. In specific embodiments, thesecondary assay system uses non-human animals, including animalspredetermined to have a disease or disorder implicating the p21 pathway,such as an angiogenic, apoptotic, or cell proliferation disorder (e.g.cancer).

The invention further provides methods for modulating the FLJ20647function and/or the p21 pathway in a mammalian cell by contacting themammalian cell with an agent that specifically binds a FLJ20647polypeptide or nucleic acid. The agent may be a small moleculemodulator, a nucleic acid modulator, or an antibody and may beadministered to a mammalian animal predetermined to have a pathologyassociated the p21 pathway.

DETAILED DESCRIPTION OF THE INVENTION

A dominant loss of function screen was carried out in Drosophila toidentify genes that interact with the cyclin dependent kinase inhibitor,p21 (Bourne H R, et al., Nature (1990) 348(6297):125-132; Marshall C J,Trends Genet (1991) 7(3):91-95). Expression of the p21 gene in the eyecauses deterioration of normal eye morphology. Modifiers of the eyephenotype were identified as members of the p21 pathway. The CG18769gene was identified as a modifier of the p21 pathway. Accordingly,vertebrate orthologs of these modifiers, and preferably the humanorthologs, FLJ20647 genes (i.e., nucleic acids and polypeptides) areattractive drug targets for the treatment of pathologies associated witha defective p21 signaling pathway, such as cancer.

In vitro and in vivo methods of assessing FLJ20647 function are providedherein. Modulation of the FLJ20647 or their respective binding partnersis useful for understanding the association of the p21 pathway and itsmembers in normal and disease conditions and for developing diagnosticsand therapeutic modalities for p21 related pathologies.FLJ20647-modulating agents that act by inhibiting or enhancing FLJ20647expression, directly or indirectly, for example, by affecting anFLJ20647 function such as binding activity, can be identified usingmethods provided herein. FLJ20647 modulating agents are useful indiagnosis, therapy and pharmaceutical development.

Nucleic Acids and Polypeptides of the Invention

Sequences related to FLJ20647 nucleic acids and polypeptides that can beused in the invention are disclosed in Genbank (referenced by Genbankidentifier (GI) number) as GI#s 20127568 (SEQ ID NO: 1) and 22051014(SEQ ID NO:2) for nucleic acid, and GI#s 8923602 (SEQ ID NO:3) and22051015 (SEQ ID NO:4) for polypeptides.

The term “FLJ20647 polypeptide” refers to a full-length FLJ20647 proteinor a functionally active fragment or derivative thereof. A “functionallyactive” FLJ20647 fragment or derivative exhibits one or more functionalactivities associated with a full-length, wild-type FLJ20647 protein,such as antigenic or immunogenic activity, ability to bind naturalcellular substrates, etc. The functional activity of FLJ20647 proteins,derivatives and fragments can be assayed by various methods known to oneskilled in the art (Current Protocols in Protein Science (1998) Coliganet al., eds., John Wiley & Sons, Inc., Somerset, N.J.) and as furtherdiscussed below. In one embodiment, a functionally active FLJ20647polypeptide is a FLJ20647 derivative capable of rescuing defectiveendogenous FLJ20647 activity, such as in cell based or animal assays;the rescuing derivative may be from the same or a different species. Forpurposes herein, functionally active fragments also include thosefragments that comprise one or more structural domains of an FLJ20647,such as a binding domain. Protein domains can be identified using thePFAM program (Bateman A., et al., Nucleic Acids Res, 1999, 27:260-2).Methods for obtaining FLJ20647 polypeptides are also further describedbelow. In some embodiments, preferred fragments are functionally active,domain-containing fragments comprising at least 25 contiguous aminoacids, preferably at least 50, more preferably 75, and most preferablyat least 100 contiguous amino acids of any one of SEQ ID NOs:3-4 (anFLJ20647). In further preferred embodiments, the fragment comprises theentire functionally active domain.

The term “FLJ20647 nucleic acid” refers to a DNA or RNA molecule thatencodes a FLJ20647 polypeptide. Preferably, the FLJ20647 polypeptide ornucleic acid or fragment thereof is from a human, but can also be anortholog, or derivative thereof with at least 70% sequence identity,preferably at least 80%, more preferably 85%, still more preferably 90%,and most preferably at least 95% sequence identity with human FLJ20647.Methods of identifying orthlogs are known in the art. Normally,orthologs in different species retain the same function, due to presenceof one or more protein motifs and/or 3-dimensional structures. Orthologsare generally identified by sequence homology analysis, such as BLASTanalysis, usually using protein bait sequences. Sequences are assignedas a potential ortholog if the best hit sequence from the forward BLASTresult retrieves the original query sequence in the reverse BLAST(Huynen M A and Bork P, Proc Natl Acad Sci (1998) 95:5849-5856; Huynen MA et al., Genome Research (2000) 10:1204-1210). Programs for multiplesequence alignment, such as CLUSTAL Thompson J D et al, 1994, NucleicAcids Res 22:46734680) may be used to highlight conserved regions and/orresidues of orthologous proteins and to generate phylogenetic trees. Ina phylogenetic tree representing multiple homologous sequences fromdiverse species (e.g., retrieved through BLAST analysis), orthologoussequences from two species generally appear closest on the tree withrespect to all other sequences from these two species. Structuralthreading or other analysis of protein folding (e.g., using software byProCeryon, Biosciences, Salzburg, Austria) may also identify potentialorthologs. In evolution, when a gene duplication event followsspeciation, a single gene in one species, such as Drosophila, maycorrespond to multiple genes (paralogs) in another, such as human. Asused herein, the term “orthologs” encompasses paralogs. As used herein,“percent (%) sequence identity” with respect to a subject sequence, or aspecified portion of a subject sequence, is defined as the percentage ofnucleotides or amino acids in the candidate derivative sequenceidentical with the nucleotides or amino acids in the subject sequence(or specified portion thereof), after aligning the sequences andintroducing gaps, if necessary to achieve the maximum percent sequenceidentity, as generated by the program WU-BLAST-2.0a19 (Altschul et al.,J. Mol. Biol. (1997) 215:403-410) with all the search parameters set todefault values. The HSP S and HSP S2 parameters are dynamic values andare established by the program itself depending upon the composition ofthe particular sequence and composition of the particular databaseagainst which the sequence of interest is being searched. A % identityvalue is determined by the number of matching identical nucleotides oramino acids divided by the sequence length for which the percentidentity is being reported. “Percent (%) amino acid sequence similarity”is determined by doing the same calculation as for determining % aminoacid sequence identity, but including conservative amino acidsubstitutions in addition to identical amino acids in the computation.

A conservative amino acid substitution is one in which an amino acid issubstituted for another amino acid having similar properties such thatthe folding or activity of the protein is not significantly affected.Aromatic amino acids that can be substituted for each other arephenylalanine, tryptophan, and tyrosine; interchangeable hydrophobicamino acids are leucine, isoleucine, methionine, and valine;interchangeable polar amino acids are glutamine and asparagine;interchangeable basic amino acids are arginine, lysine and histidine;interchangeable acidic amino acids are aspartic acid and glutamic acid;and interchangeable small amino acids are alanine, serine, threonine,cysteine and glycine.

Alternatively, an alignment for nucleic acid sequences is provided bythe local homology algorithm of Smith and Waterman (Smith and Waterman,1981, Advances in Applied Mathematics 2:482-489; database: EuropeanBioinformatics Institute; Smith and Waterman, 1981, J. of Molec. Biol.,147:195-197; Nicholas et al., 1998, “A Tutorial on Searching SequenceDatabases and Sequence Scoring Methods” (www.psc.edu) and referencescited therein.; W. R. Pearson, 1991, Genomics 11:635-650). Thisalgorithm can be applied to amino acid sequences by using the scoringmatrix developed by Dayhoff (Dayhoff: Atlas of Protein Sequences andStructure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National BiomedicalResearch Foundation, Washington, D.C., USA), and normalized by Gribskov(Gribskov 1986 Nucl. Acids Res. 14(6):6745-6763). The Smith-Watermanalgorithm may be employed where default parameters are used for scoring(for example, gap open penalty of 12, gap extension penalty of two).From the data generated, the “Match” value reflects “sequence identity.”

Derivative nucleic acid molecules of the subject nucleic acid moleculesinclude sequences that hybridize to the nucleic acid sequence of SEQ IDNOs: 1-2. The stringency of hybridization can be controlled bytemperature, ionic strength, pH, and the presence of denaturing agentssuch as formamide during hybridization and washing. Conditions routinelyused are set out in readily available procedure texts (e.g., CurrentProtocol in Molecular Biology, Vol. 1, Chap. 2.10, John Wiley & Sons,Publishers (1994); Sambrook et al., Molecular Cloning, Cold SpringHarbor (1989)). In some embodiments, a nucleic acid molecule of theinvention is capable of hybridizing to a nucleic acid moleculecontaining the nucleotide sequence of any one of SEQ ID NOs: 1-2 underhigh stringency hybridization conditions that are: prehybridization offilters containing nucleic acid for 8 hours to overnight at 65° C. in asolution comprising 6× single strength citrate (SSC) (1×SSC is 0.15 MNaCl, 0.015 M Na citrate; pH 7.0), 5×Denhardt's solution, 0.05% sodiumpyrophosphate and 100 μg/ml herring sperm DNA; hybridization for 18-20hours at 65° C. in a solution containing 6×SSC, 1× Denhardt's solution,100 μg/ml yeast tRNA and 0.05% sodium pyrophosphate; and washing offilters at 65° C. for 1 h in a solution containing 0.1×SSC and 0.1% SDS(sodium dodecyl sulfate).

In other embodiments, moderately stringent hybridization conditions areused that are: pretreatment of filters containing nucleic acid for 6 hat 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl(pH7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/mldenatured salmon sperm DNA; hybridization for 18-20 h at 40° C. in asolution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH7.5), 5 mMEDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, and10% (wt/vol) dextran sulfate; followed by washing twice for 1 hour at55° C. in a solution containing 2×SSC and 0.1% SDS.

Alternatively, low stringency conditions can be used that are:incubation for 8 hours to overnight at 37° C. in a solution comprising20% formamide, 5×SSC, 50 mM sodium phosphate (pH 7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured sheared salmonsperm DNA; hybridization in the same buffer for 18 to 20 hours; andwashing of filters in 1×SSC at about 37° C. for 1 hour.

Isolation, Production, Expression, and Mis-expression of FLJ20647Nucleic Acids and Polypeptides

FLJ20647 nucleic acids and polypeptides are useful for identifying andtesting agents that modulate FLJ20647 function and for otherapplications related to the involvement of FLJ20647 in the p21 pathway.FLJ20647 nucleic acids and derivatives and orthologs thereof may beobtained using any available method. For instance, techniques forisolating cDNA or genomic DNA sequences of interest by screening DNAlibraries or by using polymerase chain reaction (PCR) are well known inthe art. In general, the particular use for the protein will dictate theparticulars of expression, production, and purification methods. Forinstance, production of proteins for use in screening for modulatingagents may require methods that preserve specific biological activitiesof these proteins, whereas production of proteins for antibodygeneration may require structural integrity of particular epitopes.Expression of proteins to be purified for screening or antibodyproduction may require the addition of specific tags (e.g., generationof fusion proteins). Overexpression of an FLJ20647 protein for assaysused to assess FLJ20647 function, such as involvement in cell cycleregulation or hypoxic response, may require expression in eukaryoticcell lines capable of these cellular activities. Techniques for theexpression, production, and purification of proteins are well known inthe art; any suitable means therefore may be used (e.g., Higgins S J andHames B D (eds.) Protein Expression: A Practical Approach, OxfordUniversity Press Inc., New York 1999; Stanbury P F et al., Principles ofFermentation Technology, 2“ ” edition, Elsevier Science, New York, 1995;Doonan S (ed.) Protein Purification Protocols, Humana Press, New Jersey,1996; Coligan J E et al, Current Protocols in Protein Science (eds.),1999, John Wiley & Sons, New York). In particular embodiments,recombinant FLJ20647 is expressed in a cell line known to have defectivep21 function such as HCT116 colon cancer cells available from AmericanType Culture Collection (ATCC), Manassas, Va.). The recombinant cellsare used in cell-based screening assay systems of the invention, asdescribed further below.

The nucleotide sequence encoding an FLJ20647 polypeptide can be insertedinto any appropriate expression vector. The necessary transcriptionaland translational signals, including promoter/enhancer element, canderive from the native FLJ20647 gene and/or its flanking regions or canbe heterologous. A variety of host-vector expression systems may beutilized, such as mammalian cell systems infected with virus (e.g.vaccinia virus, adenovirus, etc.); insect cell systems infected withvirus (e.g. baculovirus); microorganisms such as yeast containing yeastvectors, or bacteria transformed with bacteriophage, plasmid, or cosmidDNA. An isolated host cell strain that modulates the expression of,modifies, and/or specifically processes the gene product may be used.

To detect expression of the FLJ20647 gene product, the expression vectorcan comprise a promoter operably linked to an FLJ20647 gene nucleicacid, one or more origins of replication, and, one or more selectablemarkers (e.g. thymidine kinase activity, resistance to antibiotics,etc.). Alternatively, recombinant expression vectors can be identifiedby assaying for the expression of the FLJ20647 gene product based on thephysical or functional properties of the FLJ20647 protein in in vitroassay systems (e.g. immunoassays).

The FLJ20647 protein, fragment, or derivative may be optionallyexpressed as a fusion, or chimeric protein product (i.e. it is joinedvia a peptide bond to a heterologous protein sequence of a differentprotein), for example to facilitate purification or detection. Achimeric product can be made by ligating the appropriate nucleic acidsequences encoding the desired amino acid sequences to each other usingstandard methods and expressing the chimeric product. A chimeric productmay also be made by protein synthetic techniques, e.g. by use of apeptide synthesizer (Hunkapiller et al., Nature (1984) 310:105-111).

Once a recombinant cell that expresses the FLJ20647 gene sequence isidentified, the gene product can be isolated and purified using standardmethods (e.g. ion exchange, affinity, and gel exclusion chromatography;centrifugation; differential solubility; electrophoresis).Alternatively, native FLJ20647 proteins can be purified from naturalsources, by standard methods (e.g. immunoaffinity purification). Once aprotein is obtained, it may be quantified and its activity measured byappropriate methods, such as immunoassay, bioassay, or othermeasurements of physical properties, such as crystallography.

The methods of this invention may also use cells that have beenengineered for altered expression (mis-expression) of FLJ20647 or othergenes associated with the p21 pathway. As used herein, mis-expressionencompasses ectopic expression, over-expression, under-expression, andnon-expression (e.g. by gene knock-out or blocking expression that wouldotherwise normally occur).

Genetically Modified Animals

Animal models that have been genetically modified to alter FLJ20647expression may be used in in vivo assays to test for activity of acandidate p21 modulating agent, or to further assess the role ofFLJ20647 in a p21 pathway process such as apoptosis or cellproliferation. Preferably, the altered FLJ20647 expression results in adetectable phenotype, such as decreased or increased levels of cellproliferation, angiogenesis, or apoptosis compared to control animalshaving normal FLJ20647 expression. The genetically modified animal mayadditionally have altered p21 expression (e.g. p21 knockout). Preferredgenetically modified animals are mammals such as primates, rodents(preferably mice or rats), among others. Preferred non-mammalian speciesinclude zebrafish, C. elegans, and Drosophila. Preferred geneticallymodified animals are transgenic animals having a heterologous nucleicacid sequence present as an extrachromosomal element in a portion of itscells, i.e. mosaic animals (see, for example, techniques described byJakobovits, 1994, Curr. Biol. 4:761-763.) or stably integrated into itsgerm line DNA (i.e., in the genomic sequence of most or all of itscells). Heterologous nucleic acid is introduced into the germ line ofsuch transgenic animals by genetic manipulation of, for example, embryosor embryonic stem cells of the host animal.

Methods of making transgenic animals are well-known in the art (fortransgenic mice see Brinster et al., Proc. Nat. Acad. Sci. USA 82:4438-4442 (1985), U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Lederet al., U.S. Pat. No. 4,873,191 by Wagner et al., and Hogan, B.,Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); for particle bombardment see U.S. Pat. No.4,945,050, by Sandford et al.; for transgenic Drosophila see Rubin andSpradling, Science (1982) 218:348-53 and U.S. Pat. No. 4,670,388; fortransgenic insects see Berghammer A. J. et al., A Universal Marker forTransgenic Insects (1999) Nature 402:370-371; for transgenic Zebrafishsee Lin S., Transgenic Zebrafish, Methods Mol. Biol. (2000);136:375-3830); for microinjection procedures for fish, amphibian eggsand birds see Houdebine and Chourrout, Experientia (1991) 47:897-905;for transgenic rats see Hammer et al., Cell (1990) 63:1099-1112; and forculturing of embryonic stem (ES) cells and the subsequent production oftransgenic animals by the introduction of DNA into ES cells usingmethods such as electroporation, calcium phosphate/DNA precipitation anddirect injection see, e.g., Teratocarcinomas and Embryonic Stem Cells, APractical Approach, E. J. Robertson, ed., IRL Press (1987)). Clones ofthe nonhuman transgenic animals can be produced according to availablemethods (see Wilmut, I. et al. (1997) Nature 385:810-813; and PCTInternational Publication Nos. WO 97/07668 and WO 97/07669).

In one embodiment, the transgenic animal is a “knock-out” animal havinga heterozygous or homozygous alteration in the sequence of an endogenousFLJ20647 gene that results in a decrease of FLJ20647 function,preferably such that FLJ20647 expression is undetectable orinsignificant. Knock-out animals are typically generated by homologousrecombination with a vector comprising a transgene having at least aportion of the gene to be knocked out. Typically a deletion, addition orsubstitution has been introduced into the transgene to functionallydisrupt it. The transgene can be a human gene (e.g., from a humangenomic clone) but more preferably is an ortholog of the human genederived from the transgenic host species. For example, a mouse FLJ20647gene is used to construct a homologous recombination vector suitable foraltering an endogenous FLJ20647 gene in the mouse genome. Detailedmethodologies for homologous recombination in mice are available (seeCapecchi, Science (1989) 244:1288-1292; Joyner et al., Nature (1989)338:153-156). Procedures for the production of non-rodent transgenicmammals and other animals are also available (Houdebine and Chourrout,supra; Pursel et al., Science (1989) 244:1281-1288; Simms et al.,Bio/Technology (1988) 6:179-183). In a preferred embodiment, knock-outanimals, such as mice harboring a knockout of a specific gene, may beused to produce antibodies against the human counterpart of the genethat has been knocked out (Claesson M H et al., (1994) Scan J Immunol40:257-264; Declerck P J et al., (1995) J Biol. Chem. 270:8397-400).

In another embodiment, the transgenic animal is a “knock-in” animalhaving an alteration in its genome that results in altered expression(e.g., increased (including ectopic) or decreased expression) of theFLJ20647 gene, e.g., by introduction of additional copies of FLJ20647,or by operatively inserting a regulatory sequence that provides foraltered expression of an endogenous copy of the FLJ20647 gene. Suchregulatory sequences include inducible, tissue-specific, andconstitutive promoters and enhancer elements. The knock-in can behomozygous or heterozygous.

Transgenic nonhuman animals can also be produced that contain selectedsystems allowing for regulated expression of the transgene. One exampleof such a system that may be produced is the cre/loxP recombinase systemof bacteriophage P1 (Lakso et al., PNAS (1992) 89:6232-6236; U.S. Pat.No. 4,959,317). If a cre/loxP recombinase system is used to regulateexpression of the transgene, animals containing transgenes encoding boththe Cre recombinase and a selected protein are required. Such animalscan be provided through the construction of “double” transgenic animals,e.g., by mating two transgenic animals, one containing a transgeneencoding a selected protein and the other containing a transgeneencoding a recombinase. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355; U.S. Pat. No. 5,654,182). In a preferredembodiment, both Cre-LoxP and Flp-Frt are used in the same system toregulate expression of the transgene, and for sequential deletion ofvector sequences in the same cell (Sun X et al (2000) Nat Genet25:83-6).

The genetically modified animals can be used in genetic studies tofurther elucidate the p21 pathway, as animal models of disease anddisorders implicating defective p21 function, and for in vivo testing ofcandidate therapeutic agents, such as those identified in screensdescribed below. The candidate therapeutic agents are administered to agenetically modified animal having altered FLJ20647 function andphenotypic changes are compared with appropriate control animals such asgenetically modified animals that receive placebo treatment, and/oranimals with unaltered FLJ20647 expression that receive candidatetherapeutic agent.

In addition to the above-described genetically modified animals havingaltered FLJ20647 function, animal models having defective p21 function(and otherwise normal FLJ20647 function), can be used in the methods ofthe present invention. For example, a p21 knockout mouse can be used toassess, in vivo, the activity of a candidate p21 modulating agentidentified in one of the in vitro assays described below. p21 knockoutmice are described in the literature (Umanoff H, et al., Proc Natl AcadSci USA 1995 Feb. 28; 92(5):1709-13). Preferably, the candidate p21modulating agent when administered to a model system with cellsdefective in p21 function, produces a detectable phenotypic change inthe model system indicating that the p21 function is restored, i.e., thecells exhibit normal cell cycle progression.

Modulating Agents

The invention provides methods to identify agents that interact withand/or modulate the function of FLJ20647 and/or the p21 pathway.Modulating agents identified by the methods are also part of theinvention. Such agents are useful in a variety of diagnostic andtherapeutic applications associated with the p21 pathway, as well as infurther analysis of the FLJ20647 protein and its contribution to the p21pathway. Accordingly, the invention also provides methods for modulatingthe p21 pathway comprising the step of specifically modulating FLJ20647activity by administering a FLJ20647-interacting or -modulating agent.

As used herein, an “FLJ20647-modulating agent” is any agent thatmodulates FLJ20647 function, for example, an agent that interacts withFLJ20647 to inhibit or enhance FLJ20647 activity or otherwise affectnormal FLJ20647 function. FLJ20647 function can be affected at anylevel, including transcription, protein expression, proteinlocalization, and cellular or extra-cellular activity. In a preferredembodiment, the FLJ20647-modulating agent specifically modulates thefunction of the FLJ20647. The phrases “specific modulating agent”,“specifically modulates”, etc., are used herein to refer to modulatingagents that directly bind to the FLJ20647 polypeptide or nucleic acid,and preferably inhibit, enhance, or otherwise alter, the function of theFLJ20647. These phrases also encompass modulating agents that alter theinteraction of the FLJ20647 with a binding partner, substrate, orcofactor (e.g. by binding to a binding partner of an FLJ20647, or to aprotein/binding partner complex, and altering FLJ20647 function). In afurther preferred embodiment, the FLJ20647-modulating agent is amodulator of the p21 pathway (e.g. it restores and/or upregulates p21function) and thus is also a p21-modulating agent.

Preferred FLJ20647-modulating agents include small molecule compounds;FLJ20647-interacting proteins, including antibodies and otherbiotherapeutics; and nucleic acid modulators such as antisense and RNAinhibitors. The modulating agents may be formulated in pharmaceuticalcompositions, for example, as compositions that may comprise otheractive ingredients, as in combination therapy, and/or suitable carriersor excipients. Techniques for formulation and administration of thecompounds may be found in “Remington's Pharmaceutical Sciences” MackPublishing Co., Easton, Pa., 19^(th) edition.

Small Molecule Modulators

Small molecules are often preferred to modulate function of proteinswith enzymatic function, and/or containing protein interaction domains.Chemical agents, referred to in the art as “small molecule” compoundsare typically organic, non-peptide molecules, having a molecular weightless than 10,000, preferably less than 5,000, more preferably less than1,000, and most preferably less than 500 daltons. This class ofmodulators includes chemically synthesized molecules, for instance,compounds from combinatorial chemical libraries. Synthetic compounds maybe rationally designed or identified based on known or inferredproperties of the FLJ20647 protein or may be identified by screeningcompound libraries. Alternative appropriate modulators of this class arenatural products, particularly secondary metabolites from organisms suchas plants or fungi, which can also be identified by screening compoundlibraries for FLJ20647-modulating activity. Methods for generating andobtaining compounds are well known in the art (Schreiber S L, Science(2000) 151: 1964-1969; Radmann J and Gunther J, Science (2000)151:1947-1948).

Small molecule modulators identified from screening assays, as describedbelow, can be used as lead compounds from which candidate clinicalcompounds may be designed, optimized, and synthesized. Such clinicalcompounds may have utility in treating pathologies associated with thep21 pathway. The activity of candidate small molecule modulating agentsmay be improved several-fold through iterative secondary functionalvalidation, as further described below, structure determination, andcandidate modulator modification and testing. Additionally, candidateclinical compounds are generated with specific regard to clinical andpharmacological properties. For example, the reagents may be derivatizedand re-screened using in vitro and in vivo assays to optimize activityand minimize toxicity for pharmaceutical development.

Protein Modulators

Specific FLJ20647-interacting proteins are useful in a variety ofdiagnostic and therapeutic applications related to the p21 pathway andrelated disorders, as well as in validation assays for otherFLJ20647-modulating agents. In a preferred embodiment,FLJ20647-interacting proteins affect normal FLJ20647 function, includingtranscription, protein expression, protein localization, and cellular orextra-cellular activity. In another embodiment, FLJ20647-interactingproteins are useful in detecting and providing information about thefunction of FLJ20647 proteins, as is relevant to p21 related disorders,such as cancer (e.g., for diagnostic means).

An FLJ20647-interacting protein may be endogenous, i.e. one thatnaturally interacts genetically or biochemically with an FLJ20647, suchas a member of the FLJ20647 pathway that modulates FLJ20647 expression,localization, and/or activity. FLJ20647-modulators include dominantnegative forms of FLJ20647-interacting proteins and of FLJ20647 proteinsthemselves. Yeast two-hybrid and variant screens offer preferred methodsfor identifying endogenous FLJ20647-interacting proteins (Finley, R. L.et al. (1996) in DNA Cloning-Expression Systems: A Practical Approach,eds. Glover D. & Hames B. D (Oxford University Press, Oxford, England),pp. 169-203; Fashema S F et al., Gene (2000) 250:1-14; Drees B L CurrOpin Chem Biol (1999) 3:64-70; Vidal M and Legrain P Nucleic Acids Res(1999) 27:919-29; and U.S. Pat. No. 5,928,868). Mass spectrometry is analternative preferred method for the elucidation of protein complexes(reviewed in, e.g., Pandley A and Mann M, Nature (2000) 405:837-846;Yates J R 3^(rd), Trends Genet (2000) 16:5-8).

An FLJ20647-interacting protein may be an exogenous protein, such as anFLJ20647-specific antibody or a T-cell antigen receptor (see, e.g.,Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold SpringHarbor Laboratory; Harlow and Lane (1999) Using antibodies: a laboratorymanual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press).FLJ20647 antibodies are further discussed below.

In preferred embodiments, an FLJ20647-interacting protein specificallybinds an FLJ20647 protein. In alternative preferred embodiments, anFLJ20647-modulating agent binds an FLJ20647 substrate, binding partner,or cofactor.

Antibodies

In another embodiment, the protein modulator is an FLJ20647 specificantibody agonist or antagonist. The antibodies have therapeutic anddiagnostic utilities, and can be used in screening assays to identifyFLJ20647 modulators. The antibodies can also be used in dissecting theportions of the FLJ20647 pathway responsible for various cellularresponses and in the general processing and maturation of the FLJ20647.

Antibodies that specifically bind FLJ20647 polypeptides can be generatedusing known methods. Preferably the antibody is specific to a mammalianortholog of FLJ20647 polypeptide, and more preferably, to humanFLJ20647. Antibodies may be polyclonal, monoclonal (mAbs), humanized orchimeric antibodies, single chain antibodies, Fab fragments, F(ab).sub.2fragments, fragments produced by a FAb expression library,anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments ofany of the above. Epitopes of FLJ20647 which are particularly antigeniccan be selected, for example, by routine screening of FLJ20647polypeptides for antigenicity or by applying a theoretical method forselecting antigenic regions of a protein (Hopp and Wood (1981), Proc.Natl. Acad. Sci. U.S.A. 78:3824-28; Hopp and Wood, (1983) Mol. Immunol.20:483-89; Sutcliffe et al., (1983) Science 219:660-66) to the aminoacid sequence shown in SEQ ID NOs:3-4. Monoclonal antibodies withaffinities of 10⁸ M⁻¹ preferably 10⁹ M⁻¹ to 10¹⁰ M⁻¹, or stronger can bemade by standard procedures as described (Harlow and Lane, supra; Goding(1986) Monoclonal Antibodies: Principles and Practice (2d ed) AcademicPress, New York; and U.S. Pat. Nos. 4,381,292; 4,451,570; and4,618,577). Antibodies may be generated against crude cell extracts ofFLJ20647 or substantially purified fragments thereof. If FLJ20647fragments are used, they preferably comprise at least 10, and morepreferably, at least 20 contiguous amino acids of an FLJ20647 protein.In a particular embodiment, FLJ20647-specific antigens and/or immunogensare coupled to carrier proteins that stimulate the immune response. Forexample, the subject polypeptides are covalently coupled to the keyholelimpet hemocyanin (KLH) carrier, and the conjugate is emulsified inFreund's complete adjuvant, which enhances the immune response. Anappropriate immune system such as a laboratory rabbit or mouse isimmunized according to conventional protocols.

The presence of FLJ20647-specific antibodies is assayed by anappropriate assay such as a solid phase enzyme-linked immunosorbantassay (ELISA) using immobilized corresponding FLJ20647 polypeptides.Other assays, such as radioimmunoassays or fluorescent assays might alsobe used.

Chimeric antibodies specific to FLJ20647 polypeptides can be made thatcontain different portions from different animal species. For instance,a human immunoglobulin constant region may be linked to a variableregion of a murine mAb, such that the antibody derives its biologicalactivity from the human antibody, and its binding specificity from themurine fragment. Chimeric antibodies are produced by splicing togethergenes that encode the appropriate regions from each species (Morrison etal., Proc. Natl. Acad. Sci. (1984) 81:6851-6855; Neuberger et al.,Nature (1984) 312:604-608; Takeda et al., Nature (1985) 31:452454).Humanized antibodies, which are a form of chimeric antibodies, can begenerated by grafting complementary-determining regions (CDRs) (Carlos,T. M., J. M. Harlan. 1994. Blood 84:2068-2101) of mouse antibodies intoa background of human framework regions and constant regions byrecombinant DNA technology (Riechmann L M, et al., 1988 Nature 323:323-327). Humanized antibodies contain ˜10% murine sequences and ˜90%human sequences, and thus further reduce or eliminate immunogenicity,while retaining the antibody specificities (Co MS, and Queen C. 1991Nature 351: 501-501; Morrison S L. 1992 Ann. Rev. Immun. 10:239-265).Humanized antibodies and methods of their production are well-known inthe art (U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370).

FLJ20647-specific single chain antibodies which are recombinant, singlechain polypeptides formed by linking the heavy and light chain fragmentsof the Fv regions via an amino acid bridge, can be produced by methodsknown in the art (U.S. Pat. No. 4,946,778; Bird, Science (1988)242:423-426; Huston et al., Proc. Natl. Acad. Sci. USA (1988)85:5879-5883; and Ward et al., Nature (1989) 334:544-546).

Other suitable techniques for antibody production involve in vitroexposure of lymphocytes to the antigenic polypeptides or alternativelyto selection of libraries of antibodies in phage or similar vectors(Huse et al., Science (1989) 246:1275-1281). As used herein, T-cellantigen receptors are included within the scope of antibody modulators(Harlow and Lane, 1988, supra).

The polypeptides and antibodies of the present invention may be usedwith or without modification. Frequently, antibodies will be labeled byjoining, either covalently or non-covalently, a substance that providesfor a detectable signal, or that is toxic to cells that express thetargeted protein (Menard S, et al., Int J. Biol Markers (1989)4:131-134). A wide variety of labels and conjugation techniques areknown and are reported extensively in both the scientific and patentliterature. Suitable labels include radionuclides, enzymes, substrates,cofactors, inhibitors, fluorescent moieties, fluorescent emittinglanthanide metals, chemiluminescent moieties, bioluminescent moieties,magnetic particles, and the like (U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241). Also,recombinant immunoglobulins may be produced (U.S. Pat. No. 4,816,567).Antibodies to cytoplasmic polypeptides may be delivered and reach theirtargets by conjugation with membrane-penetrating toxin proteins (U.S.Pat. No. 6,086,900).

When used therapeutically in a patient, the antibodies of the subjectinvention are typically administered parenterally, when possible at thetarget site, or intravenously. The therapeutically effective dose anddosage regimen is determined by clinical studies. Typically, the amountof antibody administered is in the range of about 0.1 mg/kg-to about 10mg/kg of patient weight. For parenteral administration, the antibodiesare formulated in a unit dosage injectable form (e.g., solution,suspension, emulsion) in association with a pharmaceutically acceptablevehicle. Such vehicles are inherently nontoxic and non-therapeutic.Examples are water, saline, Ringer's solution, dextrose solution, and 5%human serum albumin. Nonaqueous vehicles such as fixed oils, ethyloleate, or liposome carriers may also be used. The vehicle may containminor amounts of additives, such as buffers and preservatives, whichenhance isotonicity and chemical stability or otherwise enhancetherapeutic potential. The antibodies' concentrations in such vehiclesare typically in the range of about 1 mg/ml to about 10 mg/ml.Immunotherapeutic methods are further described in the literature (U.S.Pat. No. 5,859,206; WO0073469).

Specific Biotherapeutics

In a preferred embodiment, an FLJ20647-interacting protein may havebiotherapeutic applications. Biotherapeutic agents formulated inpharmaceutically acceptable carriers and dosages may be used to activateor inhibit signal transduction pathways. This modulation may beaccomplished by binding a ligand, thus inhibiting the activity of thepathway; or by binding a receptor, either to inhibit activation of, orto activate, the receptor. Alternatively, the biotherapeutic may itselfbe a ligand capable of activating or inhibiting a receptor.Biotherapeutic agents and methods of producing them are described indetail in U.S. Pat. No. 6,146,628.

When the FLJ20647 is a ligand, it may be used as a biotherapeutic agentto activate or inhibit its natural receptor. Alternatively, antibodiesagainst FLJ20647, as described in the previous section, may be used asbiotherapeutic agents.

When the FLJ20647 is a receptor, its ligand(s), antibodies to theligand(s) or the FLJ20647 itself may be used as biotherapeutics tomodulate the activity of FLJ20647 in the p21 pathway.

Nucleic Acid Modulators

Other preferred FLJ20647-modulating agents comprise nucleic acidmolecules, such as antisense oligomers or double stranded RNA (dsRNA),which generally inhibit FLJ20647 activity. Preferred nucleic acidmodulators interfere with the function of the FLJ20647 nucleic acid suchas DNA replication, transcription, translocation of the FLJ20647 RNA tothe site of protein translation, translation of protein from theFLJ20647 RNA, splicing of the FLJ20647 RNA to yield one or more mRNAspecies, or catalytic activity which may be engaged in or facilitated bythe FLJ20647 RNA.

In one embodiment, the antisense oligomer is an oligonucleotide that issufficiently complementary to an FLJ20647 mRNA to bind to and preventtranslation, preferably by binding to the 5′ untranslated region.FLJ20647-specific antisense oligonucleotides, preferably range from atleast 6 to about 200 nucleotides. In some embodiments theoligonucleotide is preferably at least 10, 15, or 20 nucleotides inlength. In other embodiments, the oligonucleotide is preferably lessthan 50, 40, or 30 nucleotides in length. The oligonucleotide can be DNAor RNA or a chimeric mixture or derivatives or modified versionsthereof, single-stranded or double-stranded. The oligonucleotide can bemodified at the base moiety, sugar moiety, or phosphate backbone. Theoligonucleotide may include other appending groups such as peptides,agents that facilitate transport across the cell membrane,hybridization-triggered cleavage agents, and intercalating agents.

In another embodiment, the antisense oligomer is a phosphothioatemorpholino oligomer (PMO). PMOs are assembled from four differentmorpholino subunits, each of which contain one of four genetic bases (A,C, G, or T) linked to a six-membered morpholine ring. Polymers of thesesubunits are joined by non-ionic phosphodiamidate intersubunit linkages.Details of how to make and use PMOs and other antisense oligomers arewell known in the art (e.g. see WO99/18193; Probst J C, AntisenseOligodeoxynucleotide and Ribozyme Design, Methods. (2000) 22(3):271-281;Summerton J, and Weller D. 1997 Antisense Nucleic Acid Drug Dev.:7:187-95; U.S. Pat. No. 5,235,033; and U.S. Pat. No. 5,378,841).

Alternative preferred FLJ20647 nucleic acid modulators aredouble-stranded RNA species mediating RNA interference (RNAi). RNAi isthe process of sequence-specific, post-transcriptional gene silencing inanimals and plants, initiated by double-stranded RNA (dsRNA) that ishomologous in sequence to the silenced gene. Methods relating to the useof RNAi to silence genes in C. elegans, Drosophila, plants, and humansare known in the art (Fire A, et al., 1998 Nature 391:806-811; Fire, A.Trends Genet. 15, 358-363 (1999); Sharp, P. A. RNA interference 2001.Genes Dev. 15, 485-490 (2001); Hammond, S. M., et al., Nature Rev.Genet. 2, 110-1119 (2001); Tuschl, T. Chem. Biochem. 2, 239-245 (2001);Hamilton, A. et al., Science 286, 950-952 (1999); Hammond, S. M., etal., Nature 404, 293-296 (2000); Zamore, P. D., et al., Cell 101, 25-33(2000); Bernstein, E., et al., Nature 409, 363-366 (2001); Elbashir, S.M., et al., Genes Dev. 15, 188-200 (2001); WO0129058; WO9932619;Elbashir S M, et al., 2001 Nature 411:494-498).

Nucleic acid modulators are commonly used as research reagents,diagnostics, and therapeutics. For example, antisense oligonucleotides,which are able to inhibit gene expression with exquisite specificity,are often used to elucidate the function of particular genes (see, forexample, U.S. Pat. No. 6,165,790). Nucleic acid modulators are alsoused, for example, to distinguish between functions of various membersof a biological pathway. For example, antisense oligomers have beenemployed as therapeutic moieties in the treatment of disease states inanimals and man and have been demonstrated in numerous clinical trialsto be safe and effective (Milligan J F, et al, Current Concepts inAntisense Drug Design, J Med. Chem. (1993) 36:1923-1937; Tonkinson J Let al., Antisense Oligodeoxynucleotides as Clinical Therapeutic Agents,Cancer Invest. (1996) 14:54-65). Accordingly, in one aspect of theinvention, an FLJ20647-specific nucleic acid modulator is used in anassay to further elucidate the role of the FLJ20647 in the p21 pathway,and/or its relationship to other members of the pathway. In anotheraspect of the invention, an FLJ20647-specific antisense oligomer is usedas a therapeutic agent for treatment of p21-related disease states.

Assay Systems

The invention provides assay systems and screening methods foridentifying specific modulators of FLJ20647 activity. As used herein, an“assay system” encompasses all the components required for performingand analyzing results of an assay that detects and/or measures aparticular event. In general, primary assays are used to identify orconfirm a modulator's specific biochemical or molecular effect withrespect to the FLJ20647 nucleic acid or protein. In general, secondaryassays further assess the activity of a FLJ20647 modulating agentidentified by a primary assay and may confirm that the modulating agentaffects FLJ20647 in a manner relevant to the p21 pathway. In some cases,FLJ20647 modulators will be directly tested in a secondary assay.

In a preferred embodiment, the screening method comprises contacting asuitable assay system comprising an FLJ20647 polypeptide or nucleic acidwith a candidate agent under conditions whereby, but for the presence ofthe agent, the system provides a reference activity (e.g. bindingactivity), which is based on the particular molecular event thescreening method detects. A statistically significant difference betweenthe agent-biased activity and the reference activity indicates that thecandidate agent modulates FLJ20647 activity, and hence the p21 pathway.The FLJ20647 polypeptide or nucleic acid used in the assay may compriseany of the nucleic acids or polypeptides described above.

Primary Assays

The type of modulator tested generally determines the type of primaryassay.

Primary Assays for Small Molecule Modulators

For small molecule modulators, screening assays are used to identifycandidate modulators. Screening assays may be cell-based or may use acell-free system that recreates or retains the relevant biochemicalreaction of the target protein (reviewed in Sittampalam G S et al., CurrOpin Chem Biol (1997) 1:384-91 and accompanying references). As usedherein the term “cell-based” refers to assays using live cells, deadcells, or a particular cellular fraction, such as a membrane,endoplasmic reticulum, or mitochondrial fraction. The term “cell free”encompasses assays using substantially purified protein (eitherendogenous or recombinantly produced), partially purified or crudecellular extracts. Screening assays may detect a variety of molecularevents, including protein-DNA interactions, protein-protein interactions(e.g., receptor-ligand binding), transcriptional activity (e.g., using areporter gene), enzymatic activity (e.g., via a property of thesubstrate), activity of second messengers, immunogenicty and changes incellular morphology or other cellular characteristics. Appropriatescreening assays may use a wide range of detection methods includingfluorescent, radioactive, calorimetric, spectrophotometric, andamperometric methods, to provide a read-out for the particular molecularevent detected.

Cell-based screening assays usually require systems for recombinantexpression of FLJ20647 and any auxiliary proteins demanded by theparticular assay. Appropriate methods for generating recombinantproteins produce sufficient quantities of proteins that retain theirrelevant biological activities and are of sufficient purity to optimizeactivity and assure assay reproducibility. Yeast two-hybrid and variantscreens, and mass spectrometry provide preferred methods for determiningprotein-protein interactions and elucidation of protein complexes. Incertain applications, when FLJ20647-interacting proteins are used inscreens to identify small molecule modulators, the binding specificityof the interacting protein to the FLJ20647 protein may be assayed byvarious known methods such as substrate processing (e.g. ability of thecandidate FLJ20647-specific binding agents to function as negativeeffectors in FLJ20647-expressing cells), binding equilibrium constants(usually at least about 10⁷ M⁻¹, preferably at least about 10⁸ M⁻¹, morepreferably at least about 10⁹ M⁻¹), and immunogenicity (e.g. ability toelicit FLJ20647 specific antibody in a heterologous host such as amouse, rat, goat or rabbit). For enzymes and receptors, binding may beassayed by, respectively, substrate and ligand processing.

The screening assay may measure a candidate agent's ability tospecifically bind to or modulate activity of a FLJ20647 polypeptide, afusion protein thereof, or to cells or membranes bearing the polypeptideor fusion protein. The FLJ20647 polypeptide can be full length or afragment thereof that retains functional FLJ20647 activity. The FLJ20647polypeptide may be fused to another polypeptide, such as a peptide tagfor detection or anchoring, or to another tag. The FLJ20647 polypeptideis preferably human FLJ20647, or is an ortholog or derivative thereof asdescribed above. In a preferred embodiment, the screening assay detectscandidate agent-based modulation of FLJ20647 interaction with a bindingtarget, such as an endogenous or exogenous protein or other substratethat has FLJ20647-specific binding activity, and can be used to assessnormal FLJ20647 gene function.

Suitable assay formats that may be adapted to screen for FLJ20647modulators are known in the art. Preferred screening assays are highthroughput or ultra high throughput and thus provide automated,cost-effective means of screening compound libraries for lead compounds(Fernandes P B, Curr Opin Chem Biol (1998) 2:597-603; Sundberg S A, CurrOpin Biotechnol 2000, 11:47-53). In one preferred embodiment, screeningassays uses fluorescence technologies, including fluorescencepolarization, time-resolved fluorescence, and fluorescence resonanceenergy transfer. These systems offer means to monitor protein-protein orDNA-protein interactions in which the intensity of the signal emittedfrom dye-labeled molecules depends upon their interactions with partnermolecules (e.g., Selvin P R, Nat Struct Biol (2000) 7:7304; Fernandes PB, supra; Hertzberg R P and Pope A J, Curr Opin Chem Biol (2000)4:445-451).

A variety of suitable assay systems may be used to identify candidateFLJ20647 and p21 pathway modulators (e.g. U.S. Pat. Nos. 5,550,019 and6,133,437 (apoptosis assays); and U.S. Pat. Nos. 5,976,782, 6,225,118and 6,444,434 (angiogenesis assays), among others). Specific preferredassays are described in more detail below.

Apoptosis assays. Assays for apoptosis may be performed by terminaldeoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick endlabeling (TUNEL) assay. The TUNEL assay is used to measure nuclear DNAfragmentation characteristic of apoptosis (Lazebnik et al., 1994, Nature371, 346), by following the incorporation of fluorescein-dUTP (Yoneharaet al., 1989, J. Exp. Med. 169, 1747). Apoptosis may further be assayedby acridine orange staining of tissue culture cells (Lucas, R., et al.,1998, Blood 15:4730-41). Other cell-based apoptosis assays include thecaspase-3/7 assay and the cell death nucleosome ELISA assay. The caspase3/7 assay is based on the activation of the caspase cleavage activity aspart of a cascade of events that occur during programmed cell death inmany apoptotic pathways. In the caspase 3/7 assay (commerciallyavailable Apo-ONE™ Homogeneous Caspase-3/7 assay from Promega, cat#67790), lysis buffer and caspase substrate are mixed and added to cells.The caspase substrate becomes fluorescent when cleaved by active caspase3/7. The nucleosome ELISA assay is a general cell death assay known tothose skilled in the art, and available commercially (Roche, Cat#1774425). This assay is a quantitative sandwich-enzyme-immunoassay whichuses monoclonal antibodies directed against DNA and histonesrespectively, thus specifically determining amount of mono- andoligonucleosomes in the cytoplasmic fraction of cell lysates. Mono andoligonucleosomes are enriched in the cytoplasm during apoptosis due tothe fact that DNA fragmentation occurs several hours before the plasmamembrane breaks down, allowing for accumalation in the cytoplasm.Nucleosomes are not present in the cytoplasmic fraction of cells thatare not undergoing apoptosis. An apoptosis assay system may comprise acell that expresses an FLJ20647, and that optionally has defective p21function (e.g. p21 is over-expressed or under-expressed relative towild-type cells). A test agent can be added to the apoptosis assaysystem and changes in induction of apoptosis relative to controls whereno test agent is added, identify candidate p21 modulating agents. Insome embodiments of the invention, an apoptosis assay may be used as asecondary assay to test a candidate p21 modulating agents that isinitially identified using a cell-free assay system. An apoptosis assaymay also be used to test whether FLJ20647 function plays a direct rolein apoptosis. For example, an apoptosis assay may be performed on cellsthat over- or under-express FLJ20647 relative to wild type cells.Differences in apoptotic response compared to wild type cells suggeststhat the FLJ20647 plays a direct role in the apoptotic response.Apoptosis assays are described further in U.S. Pat. No. 6,133,437.

Cell proliferation and cell cycle assays. Cell proliferation may beassayed via bromodeoxyuridine (BRDU) incorporation. This assayidentifies a cell population undergoing DNA synthesis by incorporationof BRDU into newly-synthesized DNA. Newly-synthesized DNA may then bedetected using an anti-BRDU antibody (Hoshino et al., 1986, Int. J.Cancer 38, 369; Campana et al., 1988, J. Immunol. Meth. 107, 79), or byother means.

Cell proliferation is also assayed via phospho-histone H3 staining,which identifies a cell population undergoing mitosis by phosphorylationof histone H3. Phosphorylation of histone H3 at serine 10 is detectedusing an antibody specfic to the phosphorylated form of the serine 10residue of histone H3. (Chadlee, D. N. 1995, J. Biol. Chem270:20098-105). Cell Proliferation may also be examined using[³H]-thymidine incorporation (Chen, J., 1996, Oncogene 13:1395-403;Jeoung, J., 1995, J. Biol. Chem. 270:18367-73). This assay allows forquantitative characterization of S-phase DNA syntheses. In this assay,cells synthesizing DNA will incorporate [³H]-thyridine into newlysynthesized DNA. Incorporation can then be measured by standardtechniques such as by counting of radioisotope in a scintillationcounter (e.g., Beckman LS 3800 liquid Scintillation Counter). Anotherproliferation assay uses the dye Alamar Blue (available from BiosourceInternational), which fluoresces when reduced in living cells andprovides an indirect measurement of cell number (Voytik-Harbin S L etal., 1998, In Vitro Cell Dev Biol Anim 34:239-46). Yet anotherproliferation assay, the MTS assay, is based on in vitro cytotoxicityassessment of industrial chemicals, and uses the soluble tetrazoliumsalt, MTS. MTS assays are commercially available, for example, thePromega CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay(Cat.# G5421).

Cell proliferation may also be assayed by colony formation in soft agar(Sambrook et al., Molecular Cloning, Cold Spring Harbor (1989)). Forexample, cells transformed with FLJ20647 are seeded in soft agar plates,and colonies are measured and counted after two weeks incubation.

Cell proliferation may also be assayed by measuring ATP levels asindicator of metabolically active cells. Such assays are commerciallyavailable, for example Cell Titer-Glo™, which is a luminescenthomogeneous assay available from Promega.

Involvement of a gene in the cell cycle may be assayed by flow cytometry(Gray J W et al. (1986) Int J Radiat Biol Relat Stud Phys Chem Med49:237-55). Cells transfected with an FLJ20647 may be stained withpropidium iodide and evaluated in a flow cytometer (available fromBecton Dickinson), which indicates accumulation of cells in differentstages of the cell cycle.

Accordingly, a cell proliferation or cell cycle assay system maycomprise a cell that expresses an FLJ20647, and that optionally hasdefective p21 function (e.g. p21 is over-expressed or under-expressedrelative to wild-type cells). A test agent can be added to the assaysystem and changes in cell proliferation or cell cycle relative tocontrols where no test agent is added, identify candidate p21 modulatingagents. In some embodiments of the invention, the cell proliferation orcell cycle assay may be used as a secondary assay to test a candidatep21 modulating agents that is initially identified using another assaysystem such as a cell-free assay system. A cell proliferation assay mayalso be used to test whether FLJ20647 function plays a direct role incell proliferation or cell cycle. For example, a cell proliferation orcell cycle assay may be performed on cells that over- or under-expressFLJ20647 relative to wild type cells. Differences in proliferation orcell cycle compared to wild type cells suggests that the FLJ20647 playsa direct role in cell proliferation or cell cycle.

Angiogenesis. Angiogenesis may be assayed using various humanendothelial cell systems, such as umbilical vein, coronary artery, ordermal cells. Suitable assays include Alamar Blue based assays(available from Biosource International) to measure proliferation;migration assays using fluorescent molecules, such as the use of BectonDickinson Falcon HTS FluoroBlock cell culture inserts to measuremigration of cells through membranes in presence or absence ofangiogenesis enhancer or suppressors; and tubule formation assays basedon the formation of tubular structures by endothelial cells on Matrigel®(Becton Dickinson). Accordingly, an angiogenesis assay system maycomprise a cell that expresses an FLJ20647, and that optionally hasdefective p21 function (e.g. p21 is over-expressed or under-expressedrelative to wild-type cells). A test agent can be added to theangiogenesis assay system and changes in angiogenesis relative tocontrols where no test agent is added, identify candidate p21 modulatingagents. In some embodiments of the invention, the angiogenesis assay maybe used as a secondary assay to test a candidate p21 modulating agentsthat is initially identified using another assay system. An angiogenesisassay may also be used to test whether FLJ20647 function plays a directrole in cell proliferation. For example, an angiogenesis assay may beperformed on cells that over- or under-express FLJ20647 relative to wildtype cells. Differences in angiogenesis compared to wild type cellssuggests that the FLJ20647 plays a direct role in angiogenesis. U.S.Pat. Nos. 5,976,782, 6,225,118 and 6,444,434, among others, describevarious angiogenesis assays.

Hypoic induction. The alpha subunit of the transcription factor, hypoxiainducible factor-1 (HIF-1), is upregulated in tumor cells followingexposure to hypoxia in vitro. Under hypoxic conditions, HIF-1 stimulatesthe expression of genes known to be important in tumour cell survival,such as those encoding glyolytic enzymes and VEGF. Induction of suchgenes by hypoxic conditions may be assayed by growing cells transfectedwith FLJ20647 in hypoxic conditions (such as with 0.1% O2, 5% CO₂, andbalance N2, generated in a Napco 7001 incubator (Precision Scientific))and normoxic conditions, followed by assessment of gene activity orexpression by Taqman®. For example, a hypoxic induction assay system maycomprise a cell that expresses an FLJ20647, and that optionally hasdefective p21 function (e.g. p21 is over-expressed or under-expressedrelative to wild-type cells). A test agent can be added to the hypoxicinduction assay system and changes in hypoxic response relative tocontrols where no test agent is added, identify candidate p21 modulatingagents. In some embodiments of the invention, the hypoxic inductionassay may be used as a secondary assay to test a candidate p21modulating agents that is initially identified using another assaysystem. A hypoxic induction assay may also be used to test whetherFLJ20647 function plays a direct role in the hypoxic response. Forexample, a hypoxic induction assay may be performed on cells that over-or under-express FLJ20647 relative to wild type cells. Differences inhypoxic response compared to wild type cells suggests that the FLJ20647plays a direct role in hypoxic induction.

Cell adhesion. Cell adhesion assays measure adhesion of cells topurified adhesion proteins, or adhesion of cells to each other, inpresence or absence of candidate modulating agents. Cell-proteinadhesion assays measure the ability of agents to modulate the adhesionof cells to purified proteins. For example, recombinant proteins areproduced, diluted to 2.5 g/mL in PBS, and used to coat the wells of amicrotiter plate. The wells used for negative control are not coated.Coated wells are then washed, blocked with 1% BSA, and washed again.Compounds are diluted to 2× final test concentration and added to theblocked, coated wells. Cells are then added to the wells, and theunbound cells are washed off. Retained cells are labeled directly on theplate by adding a membrane-permeable fluorescent dye, such ascalcein-AM, and the signal is quantified in a fluorescent microplatereader.

Cell-cell adhesion assays measure the ability of agents to modulatebinding of cell adhesion proteins with their native ligands. Theseassays use cells that naturally or recombinantly express the adhesionprotein of choice. In an exemplary assay, cells expressing the celladhesion protein are plated in wells of a multiwell plate. Cellsexpressing the ligand are labeled with a membrane-permeable fluorescentdye, such as BCECF, and allowed to adhere to the monolayers in thepresence of candidate agents. Unbound cells are washed off, and boundcells are detected using a fluorescence plate reader.

High-throughput cell adhesion assays have also been described. In onesuch assay, small molecule ligands and peptides are bound to the surfaceof microscope slides using a microarray spotter, intact cells are thencontacted with the slides, and unbound cells are washed off. In thisassay not only the binding specificity of the peptides and modulatorsagainst cell lines are determined, but also the functional cellsignaling of attached cells using immunofluorescence techniques in situon the microchip is measured (Falsey J R et al., Bioconjug Chem. 2001May-June; 12(3):346-53).

Tubulogenesis. Tubulogenesis assays monitor the ability of culturedcells, generally endothelial cells, to form tubular structures on amatrix substrate, which generally simulates the environment of theextracellular matrix. Exemplary substrates include Matrigel™ (BectonDickinson), an extract of basement membrane proteins containing laminin,collagen IV, and heparin sulfate proteoglycan, which is liquid at 4° C.and forms a solid gel at 37° C. Other suitable matrices compriseextracellular components such as collagen, fibronectin, and/or fibrin.Cells are stimulated with a pro-angiogenic stimulant, and their abilityto form tubules is detected by imaging. Tubules can generally bedetected after an overnight incubation with stimuli, but longer orshorter time frames may also be used. Tube formation assays are wellknown in the art (e.g., Jones M K et al., 1999, Nature Medicine5:1418-1423). These assays have traditionally involved stimulation withserum or with the growth factors FGF or VEGF. Serum represents anundefined source of growth factors. In a preferred embodiment, the assayis performed with cells cultured in serum free medium, in order tocontrol which process or pathway a candidate agent modulates. Moreover,we have found that different target genes respond differently tostimulation with different pro-angiogenic agents, including inflammatoryangiogenic factors such as TNF-alpa. Thus, in a further preferredembodiment, a tubulogenesis assay system comprises testing an FLJ20647'sresponse to a variety of factors, such as FGF, VEGF, phorbol myristateacetate (PMA), TNF-alpha, ephrin, etc.

Cell Migration. An invasion/migration assay (also called a migrationassay) tests the ability of cells to overcome a physical barrier and tomigrate towards pro-angiogenic signals. Migration assays are known inthe art (e.g., Paik J H et al., 2001, J Biol Chem 276:11830-11837). In atypical experimental set-up, cultured endothelial cells are seeded ontoa matrix-coated porous lamina, with pore sizes generally smaller thantypical cell size. The matrix generally simulates the environment of theextracellular matrix, as described above. The lamina is typically amembrane, such as the transwell polycarbonate membrane (Corning CostarCorporation, Cambridge, Mass.), and is generally part of an upperchamber that is in fluid contact with a lower chamber containingpro-angiogenic stimuli. Migration is generally assayed after anovernight incubation with stimuli, but longer or shorter time frames mayalso be used. Migration is assessed as the number of cells that crossedthe lamina, and may be detected by staining cells with hemotoxylinsolution (VWR Scientific, South San Francisco, Calif.), or by any othermethod for determining cell number. In another exemplary set up, cellsare fluorescently labeled and migration is detected using fluorescentreadings, for instance using the Falcon HTS FluoroBlok (BectonDickinson). While some migration is observed in the absence of stimulus,migration is greatly increased in response to pro-angiogenic factors. Asdescribed above, a preferred assay system for migration/invasion assayscomprises testing an FLJ20647's response to a variety of pro-angiogenicfactors, including tumor angiogenic and inflammatory angiogenic agents,and culturing the cells in serum free medium

Sprouting assay. A sprouting assay is a three-dimensional in vitroangiogenesis assay that uses a cell-number defined spheroid aggregationof endothelial cells (“spheroid”), embedded in a collagen gel-basedmatrix. The spheroid can serve as a starting point for the sprouting ofcapillary-like structures by invasion into the extracellular matrix(termed “cell sprouting”) and the subsequent formation of complexanastomosing networks (Korff and Augustin, 1999, J Cell Sci112:3249-58). In an exemplary experimental set-up, spheroids areprepared by pipetting 400 human umbilical vein endothelial cells intoindividual wells of a nonadhesive 96-well plates to allow overnightspheroidal aggregation (Korff and Augustin: J Cell Biol 143: 1341-52,1998). Spheroids are harvested and seeded in 900 μl of methocel-collagensolution and pipetted into individual wells of a 24 well plate to allowcollagen gel polymerization. Test agents are added after 30 min bypipetting 100 μl of 10-fold concentrated working dilution of the testsubstances on top of the gel. Plates are incubated at 37° C. for 24 h.Dishes are fixed at the end of the experimental incubation period byaddition of paraformaldehyde. Sprouting intensity of endothelial cellscan be quantitated by an automated image analysis system to determinethe cumulative sprout length per spheroid.

Primary Assays for Antibody Modulators

For antibody modulators, appropriate primary assays test is a bindingassay that tests the antibody's affinity to and specificity for theFLJ20647 protein. Methods for testing antibody affinity and specificityare well known in the art (Harlow and Lane, 1988, 1999, supra). Theenzyme-linked immunosorbant assay (ELISA) is a preferred method fordetecting FLJ20647-specific antibodies; others include FACS assays,radioimmunoassays, and fluorescent assays.

In some cases, screening assays described for small molecule modulatorsmay also be used to test antibody modulators.

Primary Assays for Nucleic Acid Modulators

For nucleic acid modulators, primary assays may test the ability of thenucleic acid modulator to inhibit or enhance FLJ20647 gene expression,preferably mRNA expression. In general, expression analysis comprisescomparing FLJ20647 expression in like populations of cells (e.g., twopools of cells that endogenously or recombinantly express FLJ20647) inthe presence and absence of the nucleic acid modulator. Methods foranalyzing mRNA and protein expression are well known in the art. Forinstance, Northern blotting, slot blotting, ribonuclease protection,quantitative RT-PCR (e.g., using the TaqMan®, PE Applied Biosystems), ormicroarray analysis may be used to confirm that FLJ20647 mRNA expressionis reduced in cells treated with the nucleic acid modulator (e.g.,Current Protocols in Molecular Biology (1994) Ausubel F M et al., eds.,John Wiley & Sons, Inc., chapter 4; Freeman W M et al., Biotechniques(1999) 26:112-125; Kallioniemi O P, Ann Med 2001, 33:142-147; Blohm D Hand Guiseppi-Elie, A Curr Opin Biotechnol 2001, 12:41-47). Proteinexpression may also be monitored. Proteins are most commonly detectedwith specific antibodies or antisera directed against either theFLJ20647 protein or specific peptides. A variety of means includingWestern blotting, ELISA, or in situ detection, are available (Harlow Eand Lane D, 1988 and 1999, supra).

In some cases, screening assays described for small molecule modulators,particularly in assay systems that involve FLJ20647 mRNA expression, mayalso be used to test nucleic acid modulators.

Secondary Assays

Secondary assays may be used to further assess the activity ofFLJ20647-modulating agent identified by any of the above methods toconfirm that the modulating agent affects FLJ20647 in a manner relevantto the p21 pathway. As used herein, FLJ20647-modulating agents encompasscandidate clinical compounds or other agents derived from previouslyidentified modulating agent. Secondary assays can also be used to testthe activity of a modulating agent on a particular genetic orbiochemical pathway or to test the specificity of the modulating agent'sinteraction with FLJ20647.

Secondary assays generally compare like populations of cells or animals(e.g., two pools of cells or animals that endogenously or recombinantlyexpress FLJ20647) in the presence and absence of the candidatemodulator. In general, such assays test whether treatment of cells oranimals with a candidate FLJ20647-modulating agent results in changes inthe p21 pathway in comparison to untreated (or mock- or placebo-treated)cells or animals. Certain assays use “sensitized genetic backgrounds”,which, as used herein, describe cells or animals engineered for alteredexpression of genes in the p21 or interacting pathways.

Cell-Based Assays

Cell based assays may use a mammalian cell line known to have defectivep21 function such as HCT116 colon cancer cells available from AmericanType Culture Collection (ATCC), Manassas, Va.). Cell based assays maydetect endogenous p21 pathway activity or may rely on recombinantexpression of p21 pathway components. Any of the aforementioned assaysmay be used in this cell-based format. Candidate modulators aretypically added to the cell media but may also be injected into cells ordelivered by any other efficacious means.

Animal Assays

A variety of non-human animal models of normal or defective p21 pathwaymay be used to test candidate FLJ20647 modulators. Models for defectivep21 pathway typically use genetically modified animals that have beenengineered to mis-express (e.g., over-express or lack expression in)genes involved in the p21 pathway. Assays generally require systemicdelivery of the candidate modulators, such as by oral administration,injection, etc.

In a preferred embodiment, p21 pathway activity is assessed bymonitoring neovascularization and angiogenesis. Animal models withdefective and normal p21 are used to test the candidate modulator'saffect on FLJ20647 in Matrigel® assays. Matrigel® is an extract ofbasement membrane proteins, and is composed primarily of laminin,collagen IV, and heparin sulfate proteoglycan. It is provided as asterile liquid at 4° C., but rapidly forms a solid gel at 37° C. LiquidMatrigel® is mixed with various angiogenic agents, such as bFGF andVEGF, or with human tumor cells which over-express the FLJ20647. Themixture is then injected subcutaneously (SC) into female athymic nudemice (Taconic, Germantown, N.Y.) to support an intense vascularresponse. Mice with Matrigel® pellets may be dosed via oral (PO),intraperitoneal (IP), or intravenous (IV) routes with the candidatemodulator. Mice are euthanized 5-12 days post-injection, and theMatrigel® pellet is harvested for hemoglobin analysis (Sigma plasmahemoglobin kit). Hemoglobin content of the gel is found to correlate thedegree of neovascularization in the gel.

In another preferred embodiment, the effect of the candidate modulatoron FLJ20647 is assessed via tumorigenicity assays. Tumor xenograftassays are known in the art (see, e.g., Ogawa K et al., 2000, Oncogene19:6043-6052). Xenografts are typically implanted SC into female athymicmice, 6-7 week old, as single cell suspensions either from apre-existing tumor or from in vitro culture. The tumors which expressthe FLJ20647 endogenously are injected in the flank, 1×10⁵ to 1×10⁷cells per mouse in a volume of 100 μL using a 27 gauge needle. Mice arethen ear tagged and tumors are measured twice weekly. Candidatemodulator treatment is initiated on the day the mean tumor weightreaches 100 mg. Candidate modulator is delivered IV, SC, IP, or PO bybolus administration. Depending upon the pharmacokinetics of each uniquecandidate modulator, dosing can be performed multiple times per day. Thetumor weight is assessed by measuring perpendicular diameters with acaliper and calculated by multiplying the measurements of diameters intwo dimensions. At the end of the experiment, the excised tumors maybeutilized for biomarker identification or further analyses. Forimmunohistochemistry staining, xenograft tumors are fixed in 4%paraformaldehyde, 0.1M phosphate, pH 7.2, for 6 hours at 4° C., immersedin 30% sucrose in PBS, and rapidly frozen in isopentane cooled withliquid nitrogen.

In another preferred embodiment, tumorogenicity is monitored using ahollow fiber assay, which is described in U.S. Pat. No. 5,698,413.Briefly, the method comprises implanting into a laboratory animal abiocompatible, semi-permeable encapsulation device containing targetcells, treating the laboratory animal with a candidate modulating agent,and evaluating the target cells for reaction to the candidate modulator.Implanted cells are generally human cells from a pre-existing tumor or atumor cell line. After an appropriate period of time, generally aroundsix days, the implanted samples are harvested for evaluation of thecandidate modulator. Tumorogenicity and modulator efficacy may beevaluated by assaying the quantity of viable cells present in themacrocapsule, which can be determined by tests known in the art, forexample, MTT dye conversion assay, neutral red dye uptake, trypan bluestaining, viable cell counts, the number of colonies formed in softagar, the capacity of the cells to recover and replicate in vitro, etc.

In another preferred embodiment, a tumorogenicity assay use a transgenicanimal, usually a mouse, carrying a dominant oncogene or tumorsuppressor gene knockout under the control of tissue specific regulatorysequences; these assays are generally referred to as transgenic tumorassays. In a preferred application, tumor development in the transgenicmodel is well characterized or is controlled. In an exemplary model, the“RIP1-Tag2” transgene, comprising the SV40 large T-antigen oncogeneunder control of the insulin gene regulatory regions is expressed inpancreatic beta cells and results in islet cell carcinomas (Hanahan D,1985, Nature 315:115-122; Parangi S et al, 1996, Proc Natl Acad Sci USA93: 2002-2007; Bergers G et al, 1999, Science 284:808-812). An“angiogenic switch,” occurs at approximately five weeks, as normallyquiescent capillaries in a subset of hyperproliferative islets becomeangiogenic. The RIP1-TAG2 mice die by age 14 weeks. Candidate modulatorsmay be administered at a variety of stages, including just prior to theangiogenic switch (e.g., for a model of tumor prevention), during thegrowth of small tumors (e.g., for a model of intervention), or duringthe growth of large and/or invasive tumors (e.g., for a model ofregression). Tumorogenicity and modulator efficacy can be evaluatinglife-span extension and/or tumor characteristics, including number oftumors, tumor size, tumor morphology, vessel density, apoptotic index,etc.

Diagnostic and Therapeutic Uses

Specific FLJ20647-modulating agents are useful in a variety ofdiagnostic and therapeutic applications where disease or diseaseprognosis is related to defects in the p21 pathway, such as angiogenic,apoptotic, or cell proliferation disorders. Accordingly, the inventionalso provides methods for modulating the p21 pathway in a cell,preferably a cell pre-determined to have defective or impaired p21function (e.g. due to overexpression, underexpression, or misexpressionof p21, or due to gene mutations), comprising the step of administeringan agent to the cell that specifically modulates FLJ20647 activity.Preferably, the modulating agent produces a detectable phenotypic changein the cell indicating that the p21 function is restored. The phrase“function is restored”, and equivalents, as used herein, means that thedesired phenotype is achieved, or is brought closer to normal comparedto untreated cells. For example, with restored p21 function, cellproliferation and/or progression through cell cycle may normalize, or bebrought closer to normal relative to untreated cells. The invention alsoprovides methods for treating disorders or disease associated withimpaired p21 function by administering a therapeutically effectiveamount of an FLJ20647-modulating agent that modulates the p21 pathway.The invention further provides methods for modulating FLJ20647 functionin a cell, preferably a cell pre-determined to have defective orimpaired FLJ20647 function, by administering an FLJ20647-modulatingagent. Additionally, the invention provides a method for treatingdisorders or disease associated with impaired FLJ20647 function byadministering a therapeutically effective amount of anFLJ20647-modulating agent.

The discovery that FLJ20647 is implicated in p21 pathway provides for avariety of methods that can be employed for the diagnostic andprognostic evaluation of diseases and disorders involving defects in thep21 pathway and for the identification of subjects having apredisposition to such diseases and disorders.

Various expression analysis methods can be used to diagnose whetherFLJ20647 expression occurs in a particular sample, including Northernblotting, slot blotting, ribonuclease protection, quantitative RT-PCR,and microarray analysis. (e.g., Current Protocols in Molecular Biology(1994) Ausubel F M et al., eds., John Wiley & Sons, Inc., chapter 4;Freeman W M et al., Biotechniques (1999) 26:112-125; Kallioniemi O P,Ann Med 2001, 33:142-147; Blohm and Guiseppi-Elie, Curr Opin Biotechnol2001, 12:41-47). Tissues having a disease or disorder implicatingdefective p21 signaling that express an FLJ20647, are identified asamenable to treatment with an FLJ20647 modulating agent. In a preferredapplication, the p21 defective tissue overexpresses an FLJ20647 relativeto normal tissue. For example, a Northern blot analysis of mRNA fromtumor and normal cell lines, or from tumor and matching normal tissuesamples from the same patient, using full or partial FLJ20647 cDNAsequences as probes, can determine whether particular tumors express oroverexpress FLJ20647. Alternatively, the TaqMan® is used forquantitative RT-PCR analysis of FLJ20647 expression in cell lines,normal tissues and tumor samples (PE Applied Biosystems).

Various other diagnostic methods may be performed, for example,utilizing reagents such as the FLJ20647 oligonucleotides, and antibodiesdirected against an FLJ120647, as described above for: (1) the detectionof the presence of FLJ20647 gene mutations, or the detection of eitherover- or under-expression of FLJ20647 mRNA relative to the non-disorderstate; (2) the detection of either an over- or an under-abundance ofFLJ20647 gene product relative to the non-disorder state; and (3) thedetection of perturbations or abnormalities in the signal transductionpathway mediated by FLJ20647.

Thus, in a specific embodiment, the invention is drawn to a method fordiagnosing a disease or disorder in a patient that is associated withalterations in FLJ20647 expression, the method comprising: a) obtaininga biological sample from the patient; b) contacting the sample with aprobe for FLJ20647 expression; c) comparing results from step (b) with acontrol; and d) determining whether step (c) indicates a likelihood ofthe disease or disorder. Preferably, the disease is cancer, mostpreferably kidney cancer. The probe may be either DNA or protein,including an antibody.

EXAMPLES

The following experimental section and examples are offered by way ofillustration and not by way of limitation.

I. Drosophila p21 Screen

A dominant loss of function screen was carried out in Drosophila toidentify genes that interact with the cyclin dependent kinase inhibitor,p21 (Bourne H R, et al., Nature (1990) 348(6297): 125-132; Marshall C J,Trends Genet (1991) 7(3):91-95). Expression of the p21 gene from GMR-p21transgene (Hay, B. A., et al. (1994) Development 120:2121-2129) in theeye causes deterioration of normal eye morphology, resulting in reduced,rough eyes. Flies carrying this transgene were maintained as a stock (P1025 F, genotype: y w; P{p21-pExp-g1-w[+]Hsp70(3'UTR)-5}). Females ofthis stock were crossed to a collection of males carrying piggyBacinsertions (Fraser M et al., Virology (1985) 145:356-361). Resultingprogeny carrying both the transgene and transposons were scored for theeffect of the transposon on the eye phenotype, i.e. whether thetransposon enhanced or suppressed (or had no effect) the eye phenotype.All data was recorded and all modifiers were retested with a repeat ofthe original cross, and the retests were scored at least twice.Modifiers of the eye phenotype were identified as members of the p21pathway. CG18769 was an enhancer of the eye phenotype. Orthologs of themodifiers are referred to herein as FLJ20647.

BLAST analysis (Altschul et al., supra) was employed to identifyorthologs of Drosophila modifiers.

Various domains, signals, and functional subunits in proteins wereanalyzed using the PSORT (Nakai K., and Horton P., Trends Biochem Sci,1999, 24:34-6; Kenta Nakai, Protein sorting signals and prediction ofsubcellular localization, Adv. Protein Chem. 54, 277-344 (2000)), PFAM(Bateman A., et al., Nucleic Acids Res, 1999, 27:260-2), SMART (PontingC P, et al., SMART: identification and annotation of domains fromsignaling and extracellular protein sequences. Nucleic Acids Res. 1999Jan. 1; 27(1):229-32), TM-HMM (Erik L. L. Sonnhammer, Gunnar von Heijne,and Anders Krogh: A hidden Markov model for predicting transmembranehelices in protein sequences. In Proc. of Sixth Int. Conf. onIntelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T.Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen Menlo Park,Calif.: AAAI Press, 1998), and clust (Remm M, and SonnhammerB.Classification of transmembrane protein families in the Caenorhabditiselegans genome and identification of human orthologs. Genome Res. 2000November; 10(11):1679-89) programs.

II. High-Throughput In Vitro Fluorescence Polarization Assay

Fluorescently-labeled FLJ20647 peptide/substrate are added to each wellof a 96-well microtiter plate, along with a test agent in a test buffer(10 mM HBEPS, 10 mM NaCl, 6 mM magnesium chloride, pH 7.6). Changes influorescence polarization, determined by using a Fluorolite FPM-2Fluorescence Polarization Microtiter System (Dynatech Laboratories,Inc), relative to control values indicates the test compound is acandidate modifier of FLJ20647 activity.

III. High-Throughput In Vitro Binding Assay.

³³P-labeled FLJ20647 peptide is added in an assay buffer (100 mM KCl, 20mM HEPES pH 7.6, 1 mM MgCl₂, 1% glycerol, 0.5% NP-40, 50 mMbeta-mercaptoethanol, 1 mg/ml BSA, cocktail of protease inhibitors)along with a test agent to the wells of a Neutralite-avidin coated assayplate and incubated at 25° C. for 1 hour. Biotinylated substrate is thenadded to each well and incubated for 1 hour. Reactions are stopped bywashing with PBS, and counted in a scintillation counter. Test agentsthat cause a difference in activity relative to control without testagent are identified as candidate p21 modulating agents.

IV. Immunoprecipitations and Immunoblotting

For coprecipitation of transfected proteins, 3×10⁶ appropriaterecombinant cells containing the FLJ20647 proteins are plated on 10-cmdishes and transfected on the following day with expression constructs.The total amount of DNA is kept constant in each transfection by addingempty vector. After 24 h, cells are collected, washed once withphosphate-buffered saline and lysed for 20 min on ice in 1 ml of lysisbuffer containing 50 mM Hepes, pH 7.9, 250 mM NaCl, 20mM-glycerophosphate, 1 mM sodium orthovanadate, 5 mM p-nitrophenylphosphate, 2 mM dithiothreitol, protease inhibitors (complete, RocheMolecular Biochemicals), and 1% Nonidet P-40. Cellular debris is removedby centrifugation twice at 15,000×g for 15 min. The cell lysate isincubated with 25 μl of M2 beads (Sigma) for 2 h at 4° C. with gentlerocking.

After extensive washing with lysis buffer, proteins bound to the beadsare solubilized by boiling in SDS sample buffer, fractionated bySDS-polyacrylamide gel electrophoresis, transferred to polyvinylidenedifluoride membrane and blotted with the indicated antibodies. Thereactive bands are visualized with horseradish peroxidase coupled to theappropriate secondary antibodies and the enhanced chemiluminescence(ECL) Western blotting detection system (Amersham Pharmacia Biotech).

V. Expression Analysis

All cell lines used in the following experiments are NCI (NationalCancer Institute) lines, and are available from ATCC (American TypeCulture Collection, Manassas, Va. 20110-2209). Normal and tumor tissueswere obtained from Impath, UC Davis, Clontech, Stratagene, Ardais,Genome Collaborative, and Ambion.

TaqMan analysis was used to assess expression levels of the disclosedgenes in various samples.

RNA was extracted from each tissue sample using Qiagen (Valencia,Calif.) RNeasy kits, following manufacturer's protocols, to a finalconcentration of 50 ng/μl. Single stranded cDNA was then synthesized byreverse transcribing the RNA samples using random hexamers and 500 ng oftotal RNA per reaction, following prototol 4304965 of Applied Biosystems(Foster City, Calif.).

Primers for expression analysis using TaqMan assay (Applied Biosystems,Foster City, Calif.) were prepared according to the TaqMan protocols,and the following criteria: a) primer pairs were designed to spanintrons to eliminate genomic contamination, and b) each primer pairproduced only one product. Expression analysis was performed using a7900HT instrument.

Taqman reactions were carried out following manufacturer's protocols, in25 μl total volume for 96-well plates and 10 μl total volume for384-well plates, using 300 nM primer and 250 nM probe, and approximately25 ng of cDNA. The standard curve for result analysis was prepared usinga universal pool of human cDNA samples, which is a mixture of cDNAs froma wide variety of tissues so that the chance that a target will bepresent in appreciable amounts is good. The raw data were normalizedusing 18S rRNA (universally expressed in all tissues and cells).

For each expression analysis, tumor tissue samples were compared withmatched normal tissues from the same patient. A gene was consideredoverexpressed in a tumor when the level of expression of the gene was 2fold or higher in the tumor compared with its matched normal sample. Incases where normal tissue was not available, a universal pool of cDNAsamples was used instead. In these cases, a gene was consideredoverexpressed in a tumor sample when the difference of expression levelsbetween a tumor sample and the average of all normal samples from thesame tissue type was greater than 2 times the standard deviation of allnormal samples (i.e., Tumor−average(all normal samples)>2×STDEV(allnormal samples)).

Results indicated an overexpression in 70% of kidney tumors as comparedwith matched normal kidney samples (sample size:20). A modulatoridentified by an assay described herein can be further validated fortherapeutic effect by administration to a tumor in which the gene isoverexpressed. A decrease in tumor growth confirms therapeutic utilityof the modulator. Prior to treating a patient with the modulator, thelikelihood that the patient will respond to treatment can be diagnosedby obtaining a tumor sample from the patient, and assaying forexpression of the gene targeted by the modulator. The expression datafor the gene(s) can also be used as a diagnostic marker for diseaseprogression. The assay can be performed by expression analysis asdescribed above, by antibody directed to the gene target, or by anyother available detection method.

1. A method of identifying a candidate p21 pathway modulating agent,said method comprising the steps of: (a) providing an assay systemcomprising a FLJ20647 polypeptide or nucleic acid; (b) contacting theassay system with a test agent under conditions whereby, but for thepresence of the test agent, the system provides a reference activity;and (c) detecting a test agent-biased activity of the assay system,wherein a difference between the test agent-biased activity and thereference activity identifies the test agent as a candidate p21 pathwaymodulating agent.
 2. The method of claim 1 wherein the assay systemcomprises cultured cells that express the FLJ20647 polypeptide.
 3. Themethod of claim 2 wherein the cultured cells additionally have defectivep21 function.
 4. The method of claim 1 wherein the assay system includesa screening assay comprising a FLJ20647 polypeptide, and the candidatetest agent is a small molecule modulator.
 5. The method of claim 4wherein the assay is a binding assay.
 6. The method of claim 1 whereinthe assay system is selected from the group consisting of an apoptosisassay system, a cell proliferation assay system, an angiogenesis assaysystem, and a hypoxic induction assay system.
 7. The method of claim 1wherein the assay system includes a binding assay comprising a FLJ20647polypeptide and the candidate test agent is an antibody.
 8. The methodof claim 1 wherein the assay system includes an expression assaycomprising a FLJ20647 nucleic acid and the candidate test agent is anucleic acid modulator.
 9. The method of claim 8 wherein the nucleicacid modulator is an antisense oligomer.
 10. The method of claim 8wherein the nucleic acid modulator is a PMO.
 11. The method of claim 1additionally comprising: (d) administering the candidate p21 pathwaymodulating agent identified in (c) to a model system comprising cellsdefective in p21 function and, detecting a phenotypic change in themodel system that indicates that the p21 function is restored.
 12. Themethod of claim 11 wherein the model system is a mouse model withdefective p21 function.
 13. A method for modulating a p21 pathway of acell comprising contacting a cell defective in p21 function with acandidate modulator that specifically binds to a FLJ20647 polypeptide,whereby p21 function is restored.
 14. The method of claim 13 wherein thecandidate modulator is administered to a vertebrate animal predeterminedto have a disease or disorder resulting from a defect in p21 function.15. The method of claim 13 wherein the candidate modulator is selectedfrom the group consisting of an antibody and a small molecule.
 16. Themethod of claim 1, comprising the additional steps of: (e) providing asecondary assay system comprising cultured cells or a non-human animalexpressing FLJ20647, (f) contacting the secondary assay system with thetest agent of (b) or an agent derived therefrom under conditionswhereby, but for the presence of the test agent or agent derivedtherefrom, the system provides a reference activity; and (g) detectingan agent-biased activity of the second assay system, wherein adifference between the agent-biased activity and the reference activityof the second assay system confirms the test agent or agent derivedtherefrom as a candidate p21 pathway modulating agent, and wherein thesecond assay detects an agent-biased change in the p21 pathway.
 17. Themethod of claim 16 wherein the secondary assay system comprises culturedcells.
 18. The method of claim 16 wherein the secondary assay systemcomprises a non-human animal.
 19. The method of claim 18 wherein thenon-human animal mis-expresses a p21 pathway gene.
 20. A method ofmodulating p21 pathway in a mammalian cell comprising contacting thecell with an agent that specifically binds a FLJ20647 polypeptide ornucleic acid.
 21. The method of claim 20 wherein the agent isadministered to a mammalian animal predetermined to have a pathologyassociated with the p21 pathway.
 22. The method of claim 20 wherein theagent is a small molecule modulator, a nucleic acid modulator, or anantibody.
 23. A method for diagnosing a disease in a patient comprising:(a) obtaining a biological sample from the patient; (b) contacting thesample with a probe for FLJ20647 expression; (c) comparing results fromstep (b) with a control; (d) determining whether step (c) indicates alikelihood of disease.
 24. The method of claim 23 wherein said diseaseis cancer.
 25. The method according to claim 24, wherein said cancerkidney cancer.